Work on the role of insulin-like growth factors (IGFs) in pediatric sarcomas is continuing to focus on potential therapeutic approaches as well as further defining the downstream signaling events of IGF action. We are currently exploring both humanized moAbs directed against the IGFIR as well as small molecule inhibitors against the IGFIR kinase and are currently analyzing these reagents to determine whether they specifically inhibit metastatic behavior in several mouse models of metastasis in pediatric sarcomas. We ultimately plan to combine this treatment with inhibitors of mTOR. We have continued to study the role of mTOR signaling in pediatric sarcomas. We have linked ezrin expression and metastatic potential (see below) to mTOR activation and have reported that mTOR blockade using rapamysin and its analog, CCI 779, in rhabdomyosarcomas (RMS) and osteosarcomas inhibits both primary tumor growth and metastatic tumor growth and inhibits mTOR signaling in these tumors. We have now demonstrated that mTOR inhibition leads to AKT activation in these models, and that this activation is IGFIR driven, and can be abrogated using a humanized MoAb to the IGFIR. This finding has led to our interest in combining these agents in preclinical and hopefully clinical studies. We have recently demonstrated that one mechanism of growth inhibition by rapamycin appears to be via a HIF1alpha, VEGF inhibitory signal. We have also begun to analyze the potential additive or synergistic effects of combining standard cytotoxic chemotherapy with mTOR inhibition in vitro in RMS cell lines and have seen at least additive effects. We are plan to examine this combination in xenograft models. We have expanded our proteomic analysis of stage III RMS tumor samples from patients treated on COG protocols and expanded analysis continues to suggest that activation of mTOR signaling pathways in these tumor samples is a poor prognostic factor. This data has been presented at ASCO and will hopefully be published shortly. We plan to continue to expand these observations with larger patient numbers. Our work on the role of ezrin in Ewing's sarcomas (ES) has now shown that ezrin is very highly expressed in almost all tumors, and its high expression is associated with activation of anti-apoptotic signaling cascades. We have also demonstrated that blockade of ezrin inhibits metastatic behavior in ES xenografts in a manner analogous to OS models. We have most recently linked ezrin to beta-4 integrin signaling and have shown that blockade of the integrin also leads to inhibition of metastatic behavior in our mouse ostesosarcoma model. We have completed our study the role of chemokine signaling in metastatic behavior of OS, and demonstrated that blocking CXCR5 in OS xenografts blocks metatatic behavior. However, since this effect required pre-incubation with the chemokine antagonist, we have elected to not pursue this line of investigation. We have also begun to attempt to establish an in vitro model of tumor cell-stromal interaction using a mouse OS cell-fibroblast co-culture 3-D system. Preliminary data appears promising and our hope is to use this system to begin to screen for compounds that block this interaction.We have recently begun to a high throughput shRNAi screen in 2 human rhabdomyosarcoma cell lines. We are collaborating with Dr. Lou Staudt who has established this system. We have established the reagents necessary to perform this screen in both an alveolar and an embryonal RMS cell line, and are currently beginning to screen the library to identify genes that are required for tumor cell survival.